1) A 2-dimensional technique for native macromolecules was designed, using HPLC gel chromatography in the first dimension and Immobiline electrofocusing in the second. 2) Linear polyacrylamide solution and partially hydrolyzed agarose were tested as possible media for pore limit electrophoresis in the first dimension. 3) Agarose gel structure was found to comprise both a O-D gel (under O.9%) and a 1-D gel (above O.9%), with characteristic difference of effective gel fiber radii by one order of magnitude. 4) Buffer Electric Focusing (BEF) pH gradients consisting of 13 acids or 16 bases were computed using a moving boundary model. Predicted and experimental pH gradients were found to agree in proportion to constituent multiplicity. Transient state Joule heat production was found to require BEF at very low initial voltages, independently of leading ion concentration between O.1 and 1.O M. Steady-state pHs in BEF of amphoteric buffers were found to be isoelectric. Similarly, pIS in BEF agreed with those found in conventional electrofocusing.